Prodigiosin induces apoptosis

1. Prodigiosin from the supernatant of Serratia marcescens induces
apoptosis in haematopoietic cancer cell lines
1
Beatriz Montaner, 1
Sira Navarro, 2
Maria Pique , 3
Marta Vilaseca, 4
Marc Martinell, 4
Ernest Giralt,
2
Joan Gil & *,1
Ricardo Pe rez-Toma s
1
Departament de Biologia Cellular i Anatomia PatoloÁ gica, Unitat de Proliferacio i Diferenciacio Cellular, Universitat de Barcelona,
Barcelona, Spain; 2
Departament de CieÁ ncies FisioloÁ giques II, Unitat de BioquõÂmica, Universitat de Barcelona, Barcelona, Spain;
3
Laboratori d'Espectrometria de Masses, Universitat de Barcelona, Barcelona, Spain and 4
Departament de QuõÂmica OrgaÁ nica,
Universitat de Barcelona, Barcelona, Spain
1 The e€ects of supernatant from the bacterial strain Serratia marcescens 2170 (CS-2170) on the
viability of di€erent haematopoietic cancer cell lines (Jurkat, NSO, HL-60 and Ramos) and
nonmalignant cells (NIH-3T3 and MDCK) was studied. We examined whether this cytotoxic e€ect
was due to apoptosis, and we puri®ed the molecule responsible for this e€ect and determined its
chemical structure.
2 Using an MTT assay we showed a rapid (4 h) decrease in the number of viable cells. This
cytotoxic e€ect was due to apoptosis, according to the fragmentation pattern of DNA, Hoechst
33342 staining and FACS analysis of the phosphatidylserine externalization. This apoptosis was
blocked by using the caspase inhibitor Z-VAD.fmk, indicating the involvement of caspases.
3 Prodigiosin is a red pigment produced by various bacteria including S. marcescens. Using
mutants of S. marcescens (OF, WF and 933) that do not synthesize prodigiosin, we further showed
that prodigiosin is involved in this apoptosis. This evidence was corroborated by spectroscopic
analysis of prodigiosin isolated from S. marcescens.
4 These results indicate that prodigiosin, an immunosuppressor, induces apoptosis in
haematopoietic cancer cells with no marked toxicity in nonmalignant cells, raising the possibility
of its therapeutic use as an antineoplastic drug.
British Journal of Pharmacology (2000) 131, 585 ± 593
Keywords: Apoptosis; cancer cell lines; chemotherapy; immunosuppressor; prodigiosin
Abbreviations: cPrGHCl, cycloprodigiosin hydrochloride; ESI-MS, electrospray ionization mass spectrometry; HBC, 4-
hydroxy-2,2'-bipyrrole-5-carboxaldehyde; LPS, lipopolysaccharide; MAP, 2-methyl-3-n-amylpyrrole; MBC, 4-
methoxy-2,2'-bipyrrole-5-carboxaldehyde; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
PG, peptone glycerol; PI, propidium iodide; UP, uncedylprodigiosin; Z-VAD.fmk, N-benzyloxycarbonyl-Val-
Ala-Asp-¯uoromethyl ketone
Introduction
Apoptosis is a form of cell death in which cells actively
participate in their own destructive processes. This process is
characterized by morphological (Kerr et al., 1994) and
biochemical/molecular (Rowan & Fisher, 1997; Reed, 1999;
Wickremasinghe & Ho€brand, 1999) criteria. Cells undergoing
apoptosis shrink and lose their normal intercellular contacts
and subsequently exhibit cytoplasmic and chromatin con-
densation and internucleosomal cleavage of DNA. In the ®nal
stages, cells become fragmented into small apoptotic bodies,
which are then eliminated by phagocytosis.
Several bacterial pathogens have been identi®ed as
mediators of apoptosis in vitro and during pathogenesis
(Zychlinsky & Santonetti, 1997). These pathogens have
developed di€erent strategies to survive inside the host,
overcome natural defences and thus cause disease. Induction
of host immunosuppression by triggering apoptosis in
phagocytes, like polymorphonuclear neutrophils (PMN) and
macrophages, might represent an advantage in bacterial
invasion, since these are the most dangerous cells for bacteria.
Several pathogens like Shigella spp (Chen & Zychlinsky, 1994)
and Salmonella spp (Chen et al., 1996; Lindgren et al., 1996;
Monack et al., 1996) induce apoptosis in macrophages.
Furthermore, the induction of PMN apoptosis by Actinoba-
cillus actinomycetemcomitans has been suggested (Kato et al.,
1995).
Bacterial toxins like leukotoxin, a-toxin and hemolysin
form pores in the eukaryotic cell membrane and disrupt the
cell via osmotic swelling (Mangan et al., 1991; Hildebrand et
al., 1991; Jonas et al., 1993). Other toxins like diphtheria toxin
and exotoxin A inhibit protein synthesis, causing apoptosis in
eukaryotic cells (Morimoto & Bonavida, 1992; Kochi &
Collier, 1993). Yoshida et al. (1998) identi®ed an acidic
glycoprotein puri®ed from the crude extract of Streptococcus
pyogenes Su, which showed cell growth inhibition in vitro and
antitumour activity in vivo. Verotoxin 1, the active component
of the bacteriocin preparation from Escherichia coli, induces
apoptosis in human cancer cell lines (Arab et al., 1998) and
eliminates human astrocytoma xenografts (Arab et al., 1999).
The prevention of neoplasia by agents from bacteria that
inhibit cancer cell proliferation but are not toxic to healthy
cells is an exciting prospect. In our laboratory, during
screening of potential anticancer drugs, we observed that the
supernatant from cultures in stationary phase (growth) of the
bacterial strain S. marcescens 2170 induced death in several
cancer cell lines. On the basis of this reproducible observation
we pursued three objectives. First, we examined the e€ects of
*Author for correspondence at: Dept. Biologia Cellular i Anatomia
PatoloÁ gica, Pavello Central, 5a planta. LR 5.1, C/Feixa Llarga s/n, E
08907 L'Hospitalet (Barcelona), Spain. E-mail: rperez@bell.ub.es
British Journal of Pharmacology (2000) 131, 585 ± 593 ã 2000 Macmillan Publishers Ltd All rights reserved 0007 ± 1188/00 $15.00
www.nature.com/bjp

2. supernatant on the viability of several haematopoietic cancer
cell lines and nonmalignant cells and assessed whether this
cytotoxic e€ect was due to apoptosis. Second, we identi®ed the
molecule contained in the supernatant of S. marcescens that
was responsible for the induction of apoptosis. Third, we
puri®ed this molecule and determined its chemical structure.
Our results con®rm the molecule as an apoptotic factor with
interesting anti-cancer properties.
Methods
Chemical and reagents
Meat peptone was purchased from Difco (Detroit, MI,
U.S.A.). Glycerol was bought from Merck (Darmstadt,
Germany). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT), and propidium iodide (PI) were
purchased from Sigma Chemicals Co (St Louis, MO, U.S.A.).
N-benzyloxycarbonyl-Val-Ala-Asp-¯uoromethyl ketone (Z-
VAD.fmk) was obtained from Enzyme Systems Products
(Dublin, CA, U.S.A.). Deionized water further puri®ed with a
Millipore Milli-Q system (Bedford, MA, U.S.A.). was used.
Bacterial strains
Serratia marcescens 2170 environmental isolated is a wild type
strain that produces prodigiosin. Mutants strains of S.
marcescens-OF, -WF and -933 were de®cient in prodigiosin
biosynthesis (Mody et al., 1990). NR1 is a lipopolysaccharide
(LPS)-de®cient mutant strain of S. marcescens which does not
have the O-antigen (Palomar et al., 1993).
Bacterial cell cultures
Each bacterial strain (S. marcescens-2170, -OF, -WF, -933 and
-NR1) was inoculated into 25 ml of peptone glycerol (PG)
medium, containing 1% meat peptone and 10% glycerol in
distilled water, and cultivated for 8 h at 308C with vigorous
shaking, and then transferred to 250 ml of PG medium and
cultivated for 48 h at 308C with vigorous shaking. Bacteria
were then harvested by centrifugation at 68006g for 15 min at
48C.
Supernatants from the bacterial strains were passed through
a 0.22 mm ®lter (Nalge Nunc Int. Rochester, NY, U.S.A.) and
concentrated by a centricon1
Plus-20 (Millipore Iberica,
Barcelona, Spain). The resulting concentrated samples (named
CS-2170, CS-OF, CS-WF, CS-933 and CS-NR1) were divided
into aliquots and stored at 7208C. Protein concentration of
the CS were estimated by the Bradford method (Bradford,
1976), and used as a measure of the CS quantity added to the
growth medium of the cell lines.
Cell lines and culture conditions
Acute human T cell leukaemia cells (Jurkat clone E6-1),
myeloma cells (NSO), human promyelocytic leukaemia cells
(HL-60), human Burkitt lymphoma cells (Ramos), Swiss
mouse embryo cells (NIH-3T3) and MDCK (NBL-2)
nonmalignant canine epithelial cells were obtained from the
American Type Culture Collection (Rockville, MD, U.S.A.).
Cells were cultured in RPMI 1640 medium (Biological
Industries, Beit Haemek, Israel) except NIH-3T3, which was
cultured in DMEM medium (Biological Industries, Beit
Haemek, Israel) and MDCK, which was cultured in MEM
(Sigma). The three media were supplemented with 10% heat-
inactivated FBS, 100 u ml71
penicillin, 100 mg ml71
strepto-
mycin (all from GIBCO ± BRL, Paisley, U.K.), and 2 mM L-
glutamine (Sigma).
Cell viability assay
Cell viability was determined by the MTT assay (Mosmann,
1983). Brie¯y, 206103
cells were incubated in 96-well
microtiter cell culture plates, in the absence (control cells) or
in the presence of increasing amounts of CS-2170, CS-OF, CS-
WF, CS-933 and CS-NR1, in a ®nal volume of 100 ml. After
4 h incubation, 10 mM of MTT (diluted in PBS) was added to
each well for an additional 4 h. The blue MTT formazan
precipitate was dissolved in 100 ml of isopropanol:1N HCl
(24 : 1) and the absorbance at 550 nm was measured on a
multiwell plate reader. Cell viability was expressed as a
percentage of control. Data are shown as the mean+standard
deviation of triplicate cultures.
Analysis of DNA fragmentation
Analysis of DNA fragmentation by agarose gel electrophoresis
was performed as described previously (Bellosillo et al., 1997).
Brie¯y, 16106
cells per ml were exposured to 10 mg ml71
of
CS-2170, CS-OF, CS-WF, CS-933 and CS-NR1 and incubated
overnight (16 h). Cells were washed in PBS and resuspended in
ice-cold lysis bu€er (10 mM Tris-HCl pH 7.4, 1 mM EDTA,
0.2% Triton X-100). After incubating for 15 min at 48C, cell
lysates were centrifuged at 14,0006g for 15 min to separate
low molecular weight DNA from intact chromatin. The
supernatant was treated with 0.2 mg ml71
of proteinase K in
a bu€er containing (mM) NaCl 150, Tris-HCl 10 pH 8.0,
EDTA 40 and 1% SDS for 4 h at 378C. The DNA
preparations were phenol/chloroform extracted twice to
remove proteins. DNA was precipitated with 140 mM NaCl
and two volumes of ethanol at 7208C overnight. DNA
precipitates were recovered by centrifugation at 14,0006g for
15 min at 48C, washed twice in cool 70% ethanol and air dried.
DNA pellets were resupended in 15 ml of TE (10 mM Tris-HCl
pH 8.0, 1 mM EDTA) and treated with RNase-DNase free
(Boehringer Mannheim, Mannheim, Germany) for 1 h at
378C. 3.2 ml of loading bu€er was added to each tube and the
DNA preparations were electrophoresed in 1% agarose gels
which contained ethidium bromide. Gels were placed on a UV
light box to visualize the DNA ladder pattern.
Hoechst staining
Cell morphology was evaluated by ¯uorescence microscopy
following Hoechst 33342 DNA staining (Sigma Chemicals
Co.). Cells (46105
per ml) from each cell line were incubated
in the absence (control cells) or in the presence of CS-2170
(5 mg ml71
) and 300 nM of prodigiosin for 6 h. Cells were then
washed in PBS and resuspended in PBS plus Hoechst 33342 to
a ®nal concentration of 2 mg ml71
and incubated for 30 min at
378C in the dark. After incubation, cells were washed in PBS
and the sections were examined with a Leitz Diaplan
microscope and photographed with a Wild MPS 45 Photo-
automat system. Apoptotic cells were identi®ed by features
characteristic of apoptosis (e.g. nuclear condensation, forma-
tion of membrane blebs and apoptotic bodies).
Analysis of apoptosis by annexin V binding
Exposure of phosphatidylserine was quanti®ed by surface
annexin V-FITC (Bender MedSystems, Boehringer Man-
Prodigiosin induces apoptosis in cancer cell lines586 B. Montaner et al
British Journal of Pharmacology, vol 131 (3)

3. nheim) staining as described previously (Koopman et al., 1994;
Bellosillo et al., 1998). 46105
cells per ml were incubated for
4 h with 5 and 10 mg ml71
of CS-2170. After this, cells were
washed in PBS and resuspended in 200 ml binding bu€er (mM:
HEPES/NaOH 10 pH 7.4, NaCl 140, CaCl2 2.5) plus 0.6 ml of
annexin V-FITC Kit and incubated for 30 min at room
temperature in the dark. After incubation, we added 200 ml of
binding bu€er and propidium iodide to a ®nal concentration of
5 mg ml71
. Cells were analysed using a Becton Dickinson
FACS Calibur ¯ow cytometer (Mountain View, CA, U.S.A.).
Samples were acquired and analysed using Cell Quest software
and data were analysed with the Paint-a-gate Pro software
(Becton Dickinson).
Annexin V binds to those cells that express phosphotidyl-
serine on the outer layer of the membrane, and propidium
iodide stains the cellular DNA of those cells with a
compromised cell membrane. This allows the discrimination
of live cells (unstained with either ¯uorochrome) from early
apoptotic cells (stained only with annexin V) and advanced
apoptotic and necrotic cells (stained with both annexin V and
propidium iodide).
Puri®cation of prodigiosin
Prodigiosin was extracted by shaking the S. marcescens 2170
cells with a mixture of methanol/1N HCl (24 : 1). After
centrifugation (68006g for 15 min), the solvent of the
supernatant was evaporated under vacuum. Atmospheric
pressure liquid chromatography of the extract was performed
on silica gel with chloroform and methanol as solvents. The
eluted pigmented fractions were pooled and the chloroform/
methanol extract was vacuum evaporated, redissolved in H2O
and lyophilized. The isolated pigment was redissolved in
methanol and analysed by electrospray ionisation mass
spectrometry (ESI-MS) using a VG-Quattro triple quadrupol
mass spectrometer (Micromass, VG-Biotech, U.K.). The
isolated pigment was repuri®ed by subsequent semipreparative
HPLC carried out on a Shimadzu instrument (Kyoto, Japan).
A Nucleosil C18 reversed-phase column (25064 mm, 10 mm)
was used with a 0 ± 100% linear gradient in 30 min (A: 0.01 M
ammonium acetate, pH 7, B: 100% acetonitrile). The elution
was monitored both using diode-array UV detector (SPD-
M10AVP Shimadzu) and by ESI-MS. After repeated
injections the pooled fractions containing the major peak
were vacuum evaporated, redissolved in H2O, lyophilized and
characterized by ESI-MS and 1
H-NMR. ESI, m/z 324.4
(M+H)+
, (C20H25N3O requires 323.4381 (MW average)).
1
H-NMR (CD3OD, 500 MHz, p.p.m.); 10.71 (m, NH), 8.54
(m, NH), 7.08 (s, 1H), 6.95 (s, 1H), 6.88 (m, 1H), 6.83 (m, 1H),
6.30 (m, 1H), 6.25 (s, 1H), 3.96 (s, 3H), 2.43 (t, 2H), 1.58 (s,
3H), 1.2 ± 1.4 (m, 6H), 0.91 (t, 3H). The concentration of
prodigiosin was determined as described (Goldschmidt &
Williams, 1968).
Results
CS-2170 decreased the viability of haematopoietic cancer
cells
The e€ect of the concentrated supernatant from S. marcescens
2170 (CS-2170) on the viability of di€erent haematopoietic
cancer cell lines (Jurkat, NSO, HL-60 and Ramos) and
nonmalignant cell lines (NIH-3T3 and MDCK) was studied.
Cell lines were incubated for 4 h with several doses of CS-2170,
ranging from 1 to 10 mg ml71
, and then cell viability was
determined by the MTT assay. A dose-dependent decrease in
the number of viable cells was observed in all the cell lines
studied except NIH-3T3 and MDCK (Figure 1A). The IC50
was 1.45 mg ml71
for Jurkat, 3.03 mg ml71
for Ramos,
3.14 mg ml71
for HL-60 and between 5 and 10 mg ml71
for
NSO. We did not observe a signi®cant decrease in the viability
of the NIH-3T3 and MDCK at the doses used in the
experiment or in other nonmalignant cell lines like Swiss-
3T3. The viability study of these cell lines treated with
prodigiosin at di€erent times (4 ± 24 h) did not show
di€erences (data not shown).
CS-2170 induced apoptosis in haematopoietic cancer
cells
In order to determine whether this cytotoxic e€ect was due to
apoptosis, we analysed whether CS-2170 induces DNA
fragmentation. Agarose gel electrophoresis of DNA showed
the characteristic ladder pattern of apoptosis in all haemato-
poietic cancer cell lines incubated overnight in the presence of
10 mg ml71
of CS-2170. However, DNA laddering was not
observed in the NIH-3T3 and MDCK at this dose of CS-2170
(Figure 1B).
We corroborated these results at microscopic level using the
Hoechst 33342 staining. Fluorescence microscope allowed the
visualization of apoptotic cells with condensed or fragmented
nuclei in Jurkat and HL-60 cells but not in NIH-3T3 cells
(Figure 1C).
Furthermore, we con®rmed these results by measuring
apoptosis in the Jurkat cell line by FACS analysis with annexin
V-FITC and propidium iodide staining (Figure 2). We
incubated Jurkat cells with 0, 5 and 10 mg ml71
of CS-2170.
The mean values of the early apoptotic populations (IP7
/
Annexin V+
cells) were 4+2, 34+8 and 45+3%, respectively.
To demonstrate the involvement of caspase activation in the
apoptotic e€ect, we analysed whether the caspase inhibitor Z-
VAD.fmk prevented apoptosis (GarcõÂa-Calvo et al., 1998).
When cells were incubated with 0, 5 and 10 mg ml71
of CS-
2170 in the presence of 50 mM Z-VAD.fmk, the mean values of
the early apoptotic population were 2+0.2, 3+0.4 and 5+2%
respectively. These results show that Z-VAD.fmk inhibits the
apoptotic action of CS-2170. Furthermore, Z-VAD.fmk
blocked DNA fragmentation (data not shown).
Identi®cation from CS-2170 of the molecule responsible
of apoptosis in haematopoietic cancer cells
Samples of CS-2170 were heated to 1008C for 15 min or
subjected to di€erent protease treatments with trypsin,
proteinase K, and protease V8 in order to eliminate active
proteins from the sample. In all cases, the samples continued
inducing apoptosis (data not shown).
S. marcescens is a Gram-negative bacillus containing LPS,
which is responsible for the biological activity of endotoxin
and is located in the outer membrane of the bacteria. LPS can
be discharged from the outer membrane to the culture
medium. In order to rule out LPS as the molecule involved
in the apoptosis induced by CS-2170, we used the S.
marcescens NR1 mutant which is LPS de®cient. Both CS-
2170 and CS-NR1 induced similar DNA laddering pattern
(Figure 3), indicating that LPS is not responsible for the
apoptotic action.
Samples from mutants defective in prodigiosin biosynth-
esis (Figure 4) were examined to clarify the involvement of
this pigment in the apoptosis activity observed. Prodigiosin
(2-methyl-3-pentyl-6-methoxyprodigiosene) is synthesized in
Prodigiosin induces apoptosis in cancer cell lines 587B. Montaner et al
British Journal of Pharmacology, vol 131 (3)

4. S. marcescens by the coupling of 2-methyl-3-n-amylpyrrole
(MAP) with 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde
(MBC). Mutant OF does not form MBC, but forms 4-
hydroxy-2,2'-bipyrrole-5-carboxaldehyde (HBC) instead,
which couples with MAP to form norprodigiosin (2-
methyl-3-pentyl-6-hydroxyprodigiosene). Mutant WF does
not form HBC or MBC but forms the volatile pyrrole MAP.
Mutant 933 does not form MAP but forms MBC (Mody et
al., 1990) Figure 4. Only CS-2170 and CS-OF induced a
signi®cant decrease in the viability of Jurkat cells (Figure
5A), and the characteristic DNA ladder pattern of apoptosis
(Figure 5B). CS-WF and CS-933 decreased cell viability but
with less eciency than CS-2170 and CS-OF and with no
evidence of DNA laddering (Figure 5B). Furthermore, the
addition of puri®ed prodigiosin to CS-WF and CS-933
allowed the induction of apoptosis in Jurkat cells (data not
shown). These evidences indicate that prodigiosin and
norprodigiosin (produced by OF mutant) are involved in
this apoptosis.
Puri®cation and characterization of prodigiosin from
CS-2170 as the apoptotic factor synthesized from
S. marcescens 2170
Furthermore, the apoptotic factor was puri®ed by methanol/
HCl extraction followed by silica gel chromatography and
semipreparative reverse-phase HPLC. Electrospray ionization
mass spectrometry gave a molecular weight of 323.4, consistent
with the expected value for prodigiosin (C20H25N3O). The
structure of prodigiosin was further con®rmed by high-®eld
1
H-NMR spectroscopy. Jurkat and NIH-3T3 cell lines were
incubated with 60 to 1200 nM of prodigiosin for 4 h, and cell
viability was determined by MTT assay. Prodigiosin decreased
viability of Jurkat cells. The IC50 was higher for the NIH-3T3
cells (Figure 6A). The DNA fragmentation pattern and was
observed in Jurkat cells but not in NIH-3T3 cells (Figure 6B).
The nuclei shown a strong blue ¯uorescence and were
condensed and fragmented in Jurkat cells (Figure 6C).
Furthermore, we con®rmed these results by measuring
A B
C
Figure 1 CS-2170 induces apoptosis in haematopoietic cancer cell lines. (A) Dose-response of the e€ect of CS-2170 on cell viability.
Haematopoietic cancer cells (Jurkat, NSO, HL-60 and Ramos) and nonmalignant cell lines (MDCK and NIH-3T3) were incubated
for 4 h with 1 ± 10 mg ml71
of CS-2170. Cell viability was determined by the MTT assay as described in Methods and it is expressed
as a percentage with respect to control cells. (B) DNA fragmentation induced by CS-2170 was observed in the agarose gel
electrophoresis as described in Methods. Jurkat, NSO, HL-60, Ramos, MDCK and NIH-3T3 cells were untreated (7) or incubated
for 16 h with 10 mg ml71
of CS-2170 (+). (C) Fluorescence microscopic analysis of Jurkat, HL-60 and NIH-3T3 nuclei with
Hoechst 33342 staining. Cells were untreated (-CS-2170) or treated with 5 mg ml71
of CS-2170 for 6 h (bar=10 mm).
Prodigiosin induces apoptosis in cancer cell lines588 B. Montaner et al
British Journal of Pharmacology, vol 131 (3)

5. apoptosis in Jurkat cells by FACS analysis with annexin V-
FITC and propidium iodide staining (Figure 6D). We
incubated the cells with 0, and 300 nM of prodigiosin. The
mean values of the early apoptotic populations (IP7
/Annexin
V+
cells) were 3+0.7 and 38+2%, respectively.
Discussion
Apoptosis is involved in the action of several cancer-
chemotherapeutic agents. In the last few years, the selection
of new drugs associated with apoptosis that would be expected
to be e€ective against tumours with high proliferation like
leukemias and lymphomas has been introduced into screening
for new anticancer drugs (Cameron & Feuer, 2000). Our
®ndings demonstrate that prodigiosin released from S.
marcescens 2170 to the culture medium induced apoptosis in
four haematopoietic cancer cell lines (Jurkat, NSO, HL-60 and
Ramos) but not in nonmalignant cells (NIH-3T3 and MDCK).
Furthermore, prodigiosin is equally active in other cancer cell
lines like SW-620, DLD-1 and HGT-1 (all of gastrointestinal
origin) (Montaner et al., manuscript in preparation), and
indicates that prodigiosin may have potential as new
antineoplastic candidate.
S. marcescens is a ubiquitous bacterium inhabiting water,
soil, plants, insects and vertebrates, and it has various
characteristics including the pigment prodigiosin (Hejazi &
Falkiner, 1997). We decided to study the possible involvement
of prodigiosin in this apoptosis for di€erent reasons: (a) in our
culture conditions the supernatant from S. marcescens 2170
was always red, the characteristic colour of prodigiosin; (b)
prodigiosin should be stable at 1008C and protease-resistant
(both properties are in accordance with our previous
experiments); (c) immunosuppressive activity has been
described for prodigiosin family members (Tsuji et al., 1992;
Kawauchi et al., 1997; Songia et al., 1997; Han et al., 1998);
and (d) the immunosuppressive e€ects of prodigiosin family
members may be due to their apoptotic e€ects as Kawauchi et
al., (1997) and, recently, Azuma et al. (2000) suggested.
Figure 2 Involvement of caspases in induction of phosphatidylserine externalization by CS-2170. A representative annexin-V/IP
assay with Jurkat is shown. This assay reveals the early phosphatidylserine translocation to the surface of apoptotic cells by
incubating the cells with FITC-labelled annexin V and the latest stages of apoptotic or necrotic processes were detected with the
vital dye propidium iodide (PI). Cells staining were analysed using the Becton Dickinson FACS Calibur ¯ow cytometer as described
in Methods. (A) Control cells. (B, C) Cells were incubated for 4 h with 5 and 10 mg ml71
of CS-2170 respectively. (D) Cells were
incubated with 50 mM Z-VAD.fmk. (E, F). Cells were incubated with 5 and 10 mg ml71
of CS-2170 respectively in the presence of
50 mM Z-VAD.fmk.
Figure 3 DNA fragmentation induced by CS-2170 and CS-NR1.
Jurkat cells were untreated (7) or incubated overnight (16 h) with
10 mg ml71
of CS-2170 and CS-NR1. DNA was extracted and
subjected to agarose gel electrophoresis as described in Methods.
Prodigiosin induces apoptosis in cancer cell lines 589B. Montaner et al
British Journal of Pharmacology, vol 131 (3)

6. Prodigiosin is produced by S. marcescens, Pseudomonas
magnesiorubra, Vibrio psychroerythrus and other bacteria
(Gerber, 1975; Paruchuri & Harshey, 1987). Microscopic
observations of S. marcescens colonies showed that prodigio-
sin pigment was localized in vesicles (extracellular and cell
associated) or intracellular granules (Matsuyama et al., 1986;
Kobayashi & Ichikawa, 1991). This pigment is synthesized in a
bifurcated pathway, in which mono- and bipyrrole precursors
are synthesized separately and then coupled to form
prodigiosin (Boger & Patel, 1988). Although, we can not rule
out the possibility that CS-2170 has other minor proapoptotic
components, our results using di€erent mutant strains of S.
marcescens indicate that prodigiosin and/or norprodigiosin,
with very similar chemical structure, are involved in the
apoptotic activity (Figure 4). Identi®cation by spectroscopic
analysis of prodigiosin released from S. marcescens to the
culture medium, allow us to demonstrate that it is responsible
for the induction of apoptosis in haematopoietic cancer cells.
Recently, Han et al. (1998) described a T-cell speci®c
immunosuppression associated to prodigiosin; however, they
showed that prodigiosin did not cause signi®cant decrease in
the splenic lymphocyte viability for 24 h of incubation at
concentrations from 1 to 1000 nM, similar to our results in
nonmalignant NIH-3T3 cells (Figure 6A).
Prodigiosin 25-C (UP) and cycloprodigiosin hydrochloride
(cPrGHCl), two members of the prodigiosin family, synthe-
sized by Streptomyces spp and Pseudoalteromonas denitri®cans
respectively have been identi®ed to have immunosuppressive
activity (Tsuji et al., 1992; Kawauchi et al., 1997; Songia et al.,
1997). UP inhibits equally well both T and B human
lymphocyte proliferation, but not transformed leukaemic cell
lines (Songia et al., 1997); Songia et al. (1997) suggested that
cell-cycle related proteins such as retinoblastoma (Rb) and
cyclin-dependent Kinase-2 and -4 (Cdk-2 and Cdk-4) are the
target molecules of UP to induce growth arrest in G1 phase in
human T and B lymphocytes. Recently, Mortellaro et al.
(1999) reported that a synthetic analogue of UP, PNU156804,
has a biological e€ect indistinguishable from UP and eciently
inhibits the activation of NF-kB and AP-1 transcription
factors. Inhibition of NF-kB substantially enhances the
Figure 4 Scheme of prodigiosin biosynthesis by S. marcescens. OF, WF and 933 mutants of the prodigiosin biosynthesis are
indicated.
Figure 5 E€ect of CS from di€erent mutants in prodigiosin biosynthesis. (A) Dose-response of the e€ect of CS-2170, CS-OF, CS-
WF and CS-933 on cell viability. Jurkat cells were incubated for 4 h with 1 ± 5 mg ml71
of CS-2170, CS-OF, CS-WF and CS-933.
Cell viability was determined by the MTT assay as described in Methods. (B) DNA fragmentation induced by CS-2170, CS-OF, CS-
WF and CS-933 was analysed by agarose gel electrophoresis as described in Methods. Jurkat cells were untreated (7) or incubated
overnight with 10 mg ml71
of CS-2170, CS-OF, CS-WF and CS-933.
Prodigiosin induces apoptosis in cancer cell lines590 B. Montaner et al
British Journal of Pharmacology, vol 131 (3)

7. apoptotic potential of cancer therapies (Wang et al., 1999).
However, Kawauchi et al. (1997) and Azuma et al. (2000)
suggested that apoptosis is the mechanism of action of
cPrGHCl to induce suppression of T cell proliferation. The
molecule cPrGHCl inhibits proliferation and induces apopto-
sis in hepatocellular carcinoma cell lines, showing IC50 values
from 276 to 592 nM, compared with 8395 nM in isolated
normal rat hepatocytes, at 72 h (Yamamoto et al., 1999). Our
results show an IC50 for prodigiosin of 225 nM in Jurkat in a
shorter assay (4 h). However, the mechanisms underlying the
apoptotic e€ect of prodigiosin are unknown. cPrGHCl inhibits
vacuolar ATPase (Tsuji et al., 1992), and, like other vacuolar
ATPase inhibitors, it acidi®es the cytoplasm and apoptosis
(Gottlieb et al., 1996). The results presented in this report show
that prodigiosin-induced apoptosis is blocked by Z-VAD.fmk,
indicating that these caspases are involved in prodigiosin-
induced apoptosis in haematopoietic cancer cell lines.
Interestingly, prodigiosin induces apoptosis in Jurkat and
HL-60 cells, both of which are p53 de®cient (Chen & Haas,
1990). This evidence indicates that prodigiosin-induce apop-
A B
C D
Figure 6 Puri®ed prodigiosin induces apoptosis in Jurkat cells. (A) Dose-response of the e€ect of prodigiosin on cell viability.
Jurkat and NIH-3T3 cell lines were incubated with 60 to 1200 nM of prodigiosin for 4 h. Cell viability was determined by the MTT
assay as described in Methods and it is expressed as a percentage with respect to control cells. (B) DNA fragmentation induced by
prodigiosin. 16106
Jurkat and NIH-3T3 cells in 1 ml were untreated (7) or incubated (P) with 300 nM of prodigiosin for 16 h.
DNA was extracted and subjected to agarose gel electrophoresis as described in Methods. (C) Fluorescence microscopic analysis of
Jurkat and NIH-3T3 nuclei with Hoechst 33342 staining. Jurkat and NIH-3T3 cells were untreated (7Prodigiosin) or treated
(+Prodigiosin) with 300 nM of prodigiosin for 6 h (bar=10 mm). (D) Detection of apoptosis in Jurkat cells detected by annexin-V/
PI assay after 4 h exposure to 300 nM of prodigiosin.
Prodigiosin induces apoptosis in cancer cell lines 591B. Montaner et al
British Journal of Pharmacology, vol 131 (3)

8. tosis by a p53-independent mechanism. Oncogenesis is often
associated with defects in p53. As prodigiosin-induced
apoptosis is p53-independent, this could mean an advantage
over other chemotherapeutic drugs (Brown & Wouters, 1999;
Bunz et al., 1999).
The elucidation of the mechanisms involved in the
apoptotic action of prodigiosin and its evaluation as a possible
anticancer drug warrants further investigation.
We thank Dr P. Oru s, Dr M. Berlanga and Dr M. VinÄ as
(Microbiology Dept. University of Barcelona) for the generous gift
of bacterial strains and helpful discussions and Dr J. Llenas
(Almirall-Prodesfarma) for reviewing the manuscript and Dr M.
Dalmau and Jordi Capella for the technical assistance.
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